1. Field of Invention
This application is based on provisional patent application Ser. No. 60/097,627, filed Aug. 24, 1998.
The present invention relates to products and processes used for the detection of microbes in a sample. More specifically, the present invention provides a method and device for aiding in the detection of the presence of specific microbial contamination in food samples, clinical specimens and other products.
2. Prior Art
It is necessary to test various substances, such as foods, beverages, pharmaceuticals, cosmetics, water, and body fluids for microbial contamination, especially with certain pathogenic bacteria. Recent outbreaks of foodborne illness, implicating a variety of foods contaminated with pathogenic bacteria, such as E. coli 0157:H7, Salmonella, Listeria, Campylobacter jejuni, and Cyclospora, have underscored the need for rapid methods for microbiological analysis. Microbiological analysis is critical for assessment of safety and quality, to determine efficiency of manufacturing, and conformance with regulations.
The increased scope, significance, and need for microbiological testing served to reveal the limitations and drawbacks of conventional methods. Classical methods for determining the presence of pathogenic bacteria in samples are taking typically several days to perform. It is desired to provide rapid detection of especially pathogenic bacteria causing illnesses.
Since the desired sensitivity for most assays for pathogenic bacteria is less than one such organism in 25 grams of product, most testing methods rely on an initial enrichment step. The indigenous microflora that is usually present in many foods at high levels often interferes with the selective isolation and identification of pathogenic bacteria. Food processing such as heating, cooling, drying, freezing, addition of preservatives and other causes can sub-lethally injure bacterial cells. These injured cells are extremely sensitive to the ingredients used in selective microbiological media. Therefore, in many assays the process starts with pre-enrichment, in which the sample is incubated in a nutritious, non-selective medium to allow the resuscitation of injured or stressed bacteria. This step is followed by a selective enrichment step where the bacteria of interest are allowed to grow while the indigenous microflora is suppressed. The enrichment procedure is followed either by conventional plating methodology or a variety of more modern and rapid methods such as DNA amplification or immunoassay.
It is therefore desired to separate at an early stage the target organisms from the other flora present in the product. One such approach is the utilization of the immuno-magnetic separation technique, involving the utilization of immuno-magnetic particles specific for the target organisms. Magnetic beads with antibodies affixed to their surfaces are mixed with the sample containing the target organism. This organism will bind to the bead surfaces via the antibodies. The organism-bead complex is pulled out of the solution by a magnet, to concentrate the microorganisms.
U.S. Pat. No. 4,230,685 describes magnetically responsive microspheres having protein A associated with the outer surface. The microspheres are reacted with antibodies selective to cells, bacteria or viruses to be separated from a mixed population. The microorganism will attach to the antibody and thereby to the microspheres, and the microspheres are then used in a magnetic separation procedure. The preferred microspheres are prepared from a mixture of albumin, Protein A, and magnetic particles. The microspheres are prepared so that the Protein A is present in the exterior surface of the antibody binding. U.S. Pat. No. 4,695,393 describes a process for the preparation of such magnetic beads, which can be used in separation of microorganisms.
U.S. Pat. Nos. 5,491,068 and 5,695,946 describe a method characterized by antibody capture of the organism of interest by the application of specialized magnetic beads. It entails the incubation of the capture cells to form colonies; removal of material from the colonies with colony lift membrane; and detection of the colony material on the membrane sheet by the use of labeled antibodies, PCR or nucleic acid probes. The main problem with this method is the low sensitivity of one organism per gram. This low sensitivity is inherent in the methodology and is 50–100 fold lower than the desired sensitivity for most food pathogens.
U.S. Pat. No. 4,677,055 describes a process for concentrating bacteria utilizing magnetic gel to which anti-specific antigenic determinant antibodies are coupled. It involves the steps of obtaining medium containing the organisms possessing specific antigenic determinants and bringing them in contact with particles of the magnetic gel. This step is followed by the separation of the gel from the medium by magnetic means and inoculation into new medium.
In general there are a number of problems associated with magnetic beads. One such problem results from the small size of such beads (3–10 μm) and the large volume of the medium (250–3,000 ml). As a result it is impossible to remove the magnetic beads from such a large volume. Therefore, many procedures either use a lower sample volume (thereby reducing the sensitivity of the assay) or allow some time (8–18 hours) of pre-enrichment followed by the removal of 1–5 ml of solution for concentration of the target organisms. Another problem associated with the magnetic beads is the fact that they get coated with fat and proteins making it difficult to be collected with a magnet. The process of separating the beads from the medium and washing the unattached bacteria is labor intensive, and creates a contamination hazard of both laboratory surfaces and the beads.